EXCEED THE SPACE PROVIDED. Research plans for coming year. Specific aim 1 - Genetic basis for antigenic variation: (a) We will continue studies of the function of Borrelia sp. RecA in E. coli using in vitro mutagenesis and will start conditional gene silencing studies in transformed Borrelia cells using antisense RNA under inducible control from a shuttle vector, (b) We will continue our studies of the programming of relapses and complete a large study of the outcomes of infections in mice that are initiated with clonal populations of two isogenic serotypes, 7 and 17. (c) We will complete our mapping and sequence analysis of the archival sites for the vsp and vip genes on the Ip28 and Ip32 linear plasmids of B. hermsii with a focus on the differential features of archived alleles for serotypes that are significantly different in frequency during the first relapse. Specific aim 2 - Evolution of variable antigens: An important question about the evolution of Vsp and Vip variable antigens is relative roles of intra-species lateral gene transfer vs. recombination within single strain. We have documented the substantial contribution of intra-strain recombination in generating diversity of vsp (Rich et al., PNAS, 2001), but the origin of initial genetic novelty remains unknown. We will finish a similar analysis of the vip genes but expand on the original study by also sequencing the vip genes of a second strain (CC1) of B. hermsii and then carrying out hypothesis-driven phylogenetic and statistical analyses of the fully-sequenced HS1 strain, the new sequences CC1 strain, as well as selected orthologs of B. turicatae, B. duttonii, and B. miyamotoi s.l. Specific aim 3 - Biology of parasitism of mammalian hosts by relapsing fever spirochetes: (a) We will continue the detailed and comprehensive study of infection and immune responses of mice infected with 6. hermsii by extending the window of monitoring and sampling through the first relapse and into the second relapse and by determining whether there are inherent differences in growth rate between different serotypes. (b) We will continue to investigate the role of the nucleotide metabolism genes that are unique to relapsing fever Borrelia spp. by carrying out further complementation studies of E. coli mutants, such as purB', and by attempting insertional inactivation or anti-sense gene silencing of selected nucleotide metabolism of B. hermsii and then assessing the effect of the changes in phenotype in defined media and in mouse infections.